The objective of the present proposal is to undertake studies of the purification of human urinary erythropoietin. The techniques to be used for purification involve the use of heat treatment, affinity chromatography using immobilized Lectin and w-Amino alkyl-Sepharose, gel filtration on Sephacryl S-200 column, ion exchange chromatography, and electrofocusing. For rapid recovery and concentration of erythropoietin at different stages of purification we plan to use volatile buffers. A fluorometric assay for proteins in the nanogram range utilizing the reagent fluorescaline will be used for the protein analyses. The criteria which will be used to establish the purity of this glycoprotein will be disc-gel electrophoresis at different pH values, sodium dodecyl sulfate disc gel electrophoresis, radiolabelling of the sialic acid moiety, and immunoelectrophoresis. The fractions will be assayed at several stages of purification for protein (Fluorometric assay) and for erythropoietin using the exhypoxic polycythemia mouse assay (59Fe incorporation in red cells). The specific activity (units/mg protein) of the fractions will be determined at each stage of purification. Determination of the molecular weight, amino acid composition and identification of the sugar residues will be carried out on the purified erythropoietin preparation. Studies will also be carried out on the purified erythropoietin preparation. Studies will also be carried out on the purified erythropoietin to be assured that it is free of "colony stimulating activity" and will support erythroid colony growth in bone marrow cultures. The application of several newly developed techniques for the purification and identification of erythropoietin should provide sufficient amounts of purified erythropoietin for studies leading to a better understanding of the physicochemical characteristics, mechanism of action and therapeutic utility of this hormone.